Few studies to date have examined genetic variability of widespread tropical amphibian species over their distributional range using different kinds of molecular markers. Here, we use genetic data in an attempt to delimit evolutionary entities within two groups of Neotropical frogs, the Scinax ruber species group and the Rhinella margaritifera species group. We combined mitochondrial and nuclear markers for a phylogenetic (a total of approximately 2500 bp) and phylogeographic study (approximately 1300 bp) to test the reliability of the currently accepted taxonomic assignments and to explore the geographic structure of their genetic variation, mainly based upon samples from the French Guianan region. Phylogenetic analyses demonstrated the polyphyly of Scinax ruber and Rhinella margaritifera. S. ruber consists of six lineages that may all merit species status. Conflicting signals of mitochondrial and nuclear markers indicated, among some Scinax lineages and species, the possibility of ongoing hybridization processes. R. margaritifera consisted of 11 lineages which might represent distinct species as well. Phylogeographic analyses added further information in support of the specific status of these lineages. Lineages of low divergence were found in sympatry and were reciprocally monophyletic for mitochondrial as well as nuclear genes, indicating the existence of young lineages that should be awarded species status. Our results highlight the utility of combining phylogenetic and phylogeographic methods, as well as the use of both mitochondrial and nuclear markers within one study. This approach helped to better understand the evolutionary history of taxonomically complex groups of species. The assessment of the geographic distribution of genetic diversity in tropical amphibian communities can lead to conclusions that differ strongly from prior analyses based on the occurrence of currently recognized species alone. Such studies, therefore, hold the potential to contribute to a more objective assessment of amphibian conservation priorities in tropical areas.